TW201907011A - Method for producing galactose oligosaccharide - Google Patents

Abstract

The present invention provides a method for increasing the amount of galacto-oligosaccharides and the reaction speed according to a method for producing galactooligosaccharides. The method for producing galacto-oligosaccharides is characterized in that it is 5 to 60 mM. In the presence of sodium ions and 0.5 to 8 mM magnesium ions, β-galactosidase reacts with the substrate.

Description

Method for producing galactooligosaccharide

The present invention relates to a method for producing a galactostooligosaccharide using β-galactosidase, and more particularly, it relates to a specific metal ion at a predetermined concentration by using β-galactosidase. A method for increasing the amount of galacto-oligosaccharides produced by trisaccharide or more and the reaction speed by acting on the matrix under the coexistence of

It is well known that β-galactosidase can catalyze the hydrolysis of β-D-galactosyl bonds such as lactose and the galactosyl transfer reaction, and is used for the selective proliferation of bifidobacterium lactobacillus Manufacture of Lactose Oligosaccharides.

In the reaction using such a β-galactosidase, a method for improving the rate of galactosyl group transfer has been investigated. For example, a method has been proposed in which the transfer rate is increased by increasing the lactose concentration of the substrate and allowing β-galactosidase to react therewith (Patent Document 1).

Using the product produced by the galactosyl transfer reaction of β-galactosidase, except β-D-galactopyranosyl (1-4) β-D-galactopyranosyl-D glucose (4´ -GL) and other galacto-oligosaccharides, may also contain transglycosidated disaccharides such as β-D-galactopyryl (1-6) -D-glucose; From the viewpoint of the effect of promoting proliferation, etc., a technique for further increasing the amount of galactose oligosaccharides having a trisaccharide or more is required. From the viewpoints of reducing manufacturing costs and improving production efficiency, it is important to shorten the reaction time before the amount of galactose oligosaccharides with a trisaccharide or more is reached, and to increase the reaction speed. [Prior Art Literature] [Patent Literature]

[Patent Document 1] Japanese Patent Publication No. 5-22517

[Problems to be Solved by the Invention]

An object of the present invention is to provide a method for increasing the amount of galacto-oligosaccharides produced by a trisaccharide or more and the reaction rate in a method for producing a galacto-oligosaccharide using β-galactosidase. [Means for solving problems]

As a result of intensive studies conducted by the present inventors to solve the above-mentioned problems, it has been found that by reacting β-galactosidase with a substrate in the presence of sodium ions and magnesium ions in a specific concentration range, it is possible to increase The amount of galactose oligosaccharides produced and the reaction time before the amount of production reaches the maximum can be shortened to complete the present invention.

That is, the present invention is a method for producing galactooligosaccharides, which is characterized in that β-galactosidase and a substrate are reacted in the presence of 5 to 60 mM sodium ions and 0.5 to 8 mM magnesium ions. [Effect of the invention]

According to the manufacturing method of the present invention, the amount of galactose oligosaccharides that are more than trisaccharides can be increased, the reaction speed can be increased, and the reaction time before reaching the maximum amount can be shortened. Therefore, it is possible to efficiently produce galacto-oligosaccharides with trisaccharides or more at low cost.

[Form of Implementing Invention]

The method for producing a galacto-oligosaccharide according to the present invention is characterized in that β-galactosidase and a substrate are reacted in the presence of 5 to 60 mM sodium ions and 0.5 to 8 mM magnesium ions. Galacto-oligosaccharides are galacto-oligosaccharides of three or more sugars represented by the general formula Gal- (Gal) n-Glc (Gal represents galactose residues, Glc represents glucose, and n represents an integer of 1 to 6).

β-galactosidase is an enzyme used to catalyze a hydrolysis reaction of β-galactosyl bonds such as lactose or o-nitrophenyl-β-D-galactopyranoside, or a galactosyl transfer reaction. The β-galactosidase used in the present invention is not particularly limited. From the viewpoint of increasing the amount of galacto-oligosaccharides and the reaction rate of trisaccharide or more, it is preferably derived from Kluyveromyces Microbes of the genus, Streptcoccus, Lacobacillus, Bifidobacterium, or Bacillus, and more preferably derived from Kluyveromyces lactis, Kluyveromyces fragilis, Streptcoccus thermophilus, Lactobacillus bulgaricus, Bifidobacterium breve, especially from the genus Kluyveromyces The β-galactosidase of microorganisms is even more preferably β-galactosidase derived from Kluyveromyces lactis.

Examples of the commercially available β-galactosidase include GODO-YNL (manufactured by Contract Alcohol Co., Ltd.) derived from Kluyveromyces lactis, Maxilact LG5000 (manufactured by DSM), or a source thereof. Lactozymes 3000L (manufactured by Novozymes) of Kluyveromyces fragilis, Lactase Y-ST (manufactured by Yakult Pharmaceutical Industry Co., Ltd.) derived from Streptcoccus thermophilus, and the like.

The form of a microorganism having a β-galactosidase activity or a β-galactosidase derived from the microorganism is not particularly limited, and examples thereof include a culture solution, a strain obtained by concentrating the culture solution by centrifugation, or membrane treatment. Body concentrates or granules, dried cells, cell debris, crude enzyme solution, purified enzyme solution, enzyme powder, etc. can be prepared according to well-known methods.

For example, when using microorganisms with β-galactosidase activity, a culture liquid obtained by culturing according to a known microorganism culture method may be directly used, or a known centrifugation, membrane treatment, drying, and pulverization treatment may be performed as required, and Used as bacterial cell concentrate or granules, dried bacterial cells, bacterial cell debris liquid, etc. The bacterial cells may be in a state of living bacterial cells, or they may be subjected to an organic solvent treatment, a freeze-drying treatment, or the like to cause the bacterial cells to die.

In addition, when β-galactosidase derived from a microorganism having β-galactosidase activity is used, the purification conditions and degree of purification are not particularly limited, and general purification methods can be used. After the microorganism is cultured according to a well-known method, the cells are separated by separation means such as centrifugation and membrane treatment. If the culture supernatant contains β-galactosidase, it can be recovered to form a crude enzyme solution. In addition, if β-galactosidase is contained in the bacteria, the bacteria can be physically pulverized by a homogenizer or ultrasonic treatment, or treated in an enzyme manner by using a cell wall to dissolve an enzyme, etc. A crude enzyme solution is formed. These crude enzyme solutions can also be combined with ammonium sulfate salting-out treatment, dialysis, gel filtration chromatography, ion exchange chromatography, adsorption chromatography, affinity chromatography, etc. to form a highly purified purification. Enzyme solution.

The matrix that interacts with the above-mentioned β-galactosidase includes the following two cases: a separate matrix that functions as a galactosyl acceptor and donor, and a galactosyl acceptor and donor Coexistence. Examples of the matrix of the galactosyl group donor include lactose, o-nitrophenyl-β-D-galactopyranoside, and the like. Examples of the matrix of the galactosyl receptor include lactose, galactooligosaccharide, glucose, and glycerol.

The concentration of the substrate is appropriately set according to the type and the like. For example, when lactose is used, the concentration of the galacto-oligosaccharide and the production rate of the trisaccharide and the like are preferably 5 to 65% by mass, more preferably. It is 15 to 60 mass%. In addition, the addition amount of β-galactosidase can be appropriately adjusted according to the required reaction time, and it is preferably 10 to 1000 U, more preferably 30 to 800 U per 1 g of lactose. The reaction temperature and the like can be appropriately set depending on the optimum temperature of the β-galactosidase used and the like. For example, when β-galactosidase derived from Kluyveromyces lactis is used, the reaction temperature is preferably 30 to 50 ° C, and more preferably 40 to 50 ° C. In addition, the measurement of enzyme activity (U) was as follows: [Measurement method of β-galactosidase enzyme activity (U)] 0.5 mL of a diluted enzyme sample was taken into a test tube, and 100 mM KH ₂ PO ₄ containing manganese chloride was added. -NaOH buffer solution (pH 6.5, hereinafter referred to as "buffer") 0.5 mL, mixed to make 0.1 mM, and then incubated at 37 ° C for 3 minutes. 1.0 mL of a 0.1% solution of o-nitrophenyl-β-D-galactopyranoside (hereinafter referred to as "ONPG"), which was previously incubated at 37 ° C, was added and quickly mixed, and the temperature was accurately maintained at 37 ° C for 1 minute. The reaction was stopped by adding 2.0 mL of a 0.2 M sodium carbonate solution and rapidly mixing the mixture. In addition, 0.5 mL of the diluted enzyme sample was taken into a test tube, and 0.5 mL of a buffer solution was added and mixed. Then, 2.0 mL of a 0.2 M sodium carbonate solution was added, and the mixture was incubated at 37 ° C for 3 minutes. Then, 0.1 mL of an ONPG solution which was previously maintained at 37 ° C was added. They were mixed and kept at 37 ° C for 1 minute (blind inspection system). The absorbance at 420 nm of the test system and the blind test system was measured using distilled water as a control group, and the enzyme activity (U) was calculated according to the following formula. [Equation 1] Enzyme activity ^* = (A ₁ -A ₂ ) × 10 × B A ₁ : absorbance of test system A ₂ : absorbance of blind test system B: dilution ratio * U / ml

In the present invention, the β-galactosidase is reacted with a substrate in the presence of sodium ions and magnesium ions. The concentration of sodium ions in the reaction system is 5 to 60 mM. On the other hand, the concentration of magnesium ions is 0.5 to 8 mM, and more preferably 1.5 to 8 mM. When the sodium ion concentration is more than 60 mM or the magnesium ion concentration is more than 8 mM, the burden on desalting and purifying the obtained galactooligosaccharide becomes large and unfavorable. The coexistence of sodium ions and magnesium ions in this range can increase the amount of galactose oligosaccharides and the reaction speed of trisaccharides or more. For sodium and magnesium ions, salts such as chlorides, carbonates, acetates, and phosphates can be added to the reaction system in the form of solids or buffers. Since the pH is less likely to change after the addition, chlorination is preferred. Sodium and magnesium chloride.

Generally speaking, the galactosyl transfer reaction of β-galactosidase competes with the hydrolysis reaction of the substrate. Therefore, if β-galactosidase is applied to the substrate, the desired galactooligosaccharide is produced. At the same time, monosaccharides such as glucose or galactose are also produced due to competing hydrolysis reactions, and galactooligosaccharides that are once produced are also hydrolyzed. In this way, the galactosyl transfer reaction and the hydrolysis reaction compete with each other, and the galactosyl donor and the acceptor may also form various combinations. Therefore, it is not easy to make the galactosyl group between the specific donor and the acceptor. The transfer reaction proceeds preferentially and is not easily controlled to produce the desired galactooligosaccharide. In contrast, in the present invention, by causing β-galactosidase to act on a substrate in the presence of sodium ions and magnesium ions in a specific concentration range, galactose and oligosaccharides in particular can be used in galactose oligosaccharides. Increasing the amount of oligosaccharides produced can even increase the reaction speed and shorten the time to reach the maximum amount of oligosaccharides, and it is possible to produce galactose oligosaccharides with trisaccharides or more efficiently at low cost.

In addition, the method of the present invention is also applicable to a secondary reaction in the production of galactooligosaccharides based on a sequential reaction in which two kinds of β-galactosidases act. That is, as a primary reaction, a microorganism having β-galactosidase activity or a β-galactosidase derived from the microorganism is reacted with the substrate, and a secondary reaction is performed to make β different from the user of the primary reaction. -Galactosidase, which acts on a primary reaction solution in the presence of 5 to 60 mM sodium ions and 0.5 to 8 mM magnesium ions, can reduce unreacted substrates and increase the amount of galacto-oligosaccharides produced.

As for the microorganism having β-galactosidase activity used in a single reaction, for example, microorganisms belonging to the genus Sporobolomyces, Aspergillus, and Bacillus, particularly Microorganisms belonging to the genus Trichoderma are preferred. From the standpoint of increasing the amount of galactose oligosaccharides and the reaction speed of trisaccharides or more, Sporobolomyces singularis is even more preferred.

The form of the microorganism having β-galactosidase activity or β-galactosidase derived from the microorganism used in the primary reaction is not particularly limited, and examples thereof include a culture solution, a culture solution subjected to centrifugation, or a membrane Concentrated bacterial cells or granules, dried bacterial cells, bacterial cell debris, crude enzyme solution, purified enzyme solution, enzyme powder, etc., which are concentrated by treatment, can be prepared according to well-known methods.

For example, when using microorganisms with β-galactosidase activity, a culture liquid obtained by culturing according to a known microorganism culture method may be directly used, or a known centrifugation, membrane treatment, drying, and pulverization treatment may be performed as required, and Used as bacterial cell concentrate or granules, dried bacterial cells, bacterial cell debris liquid, etc. The bacterial cells may be in a state of living bacterial cells, or they may be subjected to an organic solvent treatment, a freeze-drying treatment, or the like to cause the bacterial cells to die.

In addition, when β-galactosidase derived from a microorganism having β-galactosidase activity is used, the purification conditions and degree of purification are not particularly limited, and general purification methods can be used. After the microorganism is cultured according to a well-known method, the cells are separated by separation means such as centrifugation and membrane treatment. If the culture supernatant contains β-galactosidase, it can be recovered to form a crude enzyme solution. In addition, if β-galactosidase is contained in the bacteria, the bacteria can be physically pulverized by a homogenizer or ultrasonic treatment, or treated in an enzyme manner by using a cell wall to dissolve an enzyme, etc., to obtain an extract in the bacteria. A crude enzyme solution is formed. These crude enzyme solutions can also be combined with ammonium sulfate salting-out treatment, dialysis, gel filtration chromatography, ion exchange chromatography, adsorption chromatography, affinity chromatography, etc. to form a highly purified purification. Enzyme solution.

In one reaction, the microorganism having β-galactosidase activity or β-galactosidase derived from the microorganism is reacted with a substrate such as lactose. The reaction conditions can be appropriately set according to the characteristics of the microorganism having β-galactosidase activity or β-galactosidase derived from the microorganism. For example, when using Trichosporum sp. As a microorganism having β-galactosidase activity and using lactose as a substrate, the concentration of lactose is preferably 10 in terms of the effect of increasing the amount and rate of galactooligosaccharide production. -60 mass%, more preferably 40-50 mass%. In addition, the addition amount of Trichoderma sp. Yeast is preferably 0.03 to 0.3U, more preferably 0.2 to 0.3U per gram of lactose. The reaction temperature is about 30 to 70 ° C, and the reaction can be performed for about 24 to 96 hours.

In the secondary reaction, in the presence of sodium and magnesium ions in a specific concentration range, β-galactosidase, which is different from the user of the primary reaction, acts on the primary reaction solution obtained in the primary reaction.

The β-galactosidase used in the secondary reaction is not particularly limited. From the viewpoint of increasing the amount of galacto-oligosaccharides and the reaction speed of trisaccharide or more, it is preferably derived from the genus Kluyveromyces , Streptcoccus, Lactobacillus, Bifidobacterium, or Bacillus genus, preferably Kluyveromyces lactis, fragile Kluyveromyces fragilis, Streptcoccus thermophilus, Lactobacillus bulgaricus, Bifidobacterium breve, especially from microorganisms belonging to the genus Kluyveromyces Β-galactosidase is even more preferably β-galactosidase derived from Kluyveromyces lactis.

By causing the β-galactosidase to act on the primary reaction solution in the presence of sodium ions and magnesium ions in a specific concentration range, it is possible to increase the amount of galactose oligosaccharides produced by a trisaccharide or more. In addition, the reaction speed can be increased, and the reaction time before the amount of trisaccharide or more galacto-oligosaccharides reaches the maximum can be shortened. The concentration of sodium ions in the secondary reaction solution is 5 to 60 mM. On the other hand, the concentration of magnesium ions is 0.5 to 8 mM, and more preferably 1.5 to 8 mM. When the sodium ion concentration is more than 60 mM or the magnesium ion concentration is more than 8 mM, the burden on desalting and purifying the obtained galactooligosaccharide becomes large and unfavorable. By allowing sodium ions and magnesium ions to exist in this concentration range, the amount of galactose oligosaccharides produced and production efficiency can be improved. For sodium and magnesium ions, salts such as chlorides, carbonates, acetates, and phosphates can be added to the reaction system in the form of solids or buffers. Since the pH is less likely to change after the addition, chlorination is preferred. Sodium and magnesium chloride.

The concentration of residual lactose in the primary reaction solution is preferably from 5 to 65% by mass, and more preferably from 15 to 60% by mass, in terms of the effect of increasing the amount of galactose oligosaccharide produced by the trisaccharide or more and the reaction speed. The addition amount of β-galactosidase is preferably 10 to 1000 U, more preferably 30 to 800 U per 1 g of residual lactose. The reaction temperature and the like can be appropriately set depending on the optimum temperature of the β-galactosidase used and the like. For example, when β-galactosidase derived from Kluyveromyces lactis is used, the reaction temperature is preferably from 30 to 50 ° C in terms of the effect of increasing the amount of galactose oligosaccharides and the rate of production, More preferably, it is 40 to 50 ° C.

As mentioned above, the reaction liquid with galactose oligosaccharide can be directly or suitably decolorized with activated carbon, filtered with diatomaceous earth, desalted with ion exchange resin, concentrated using a concentrator to make liquid sugar, Alternatively, it can be powdered by a spray dryer or the like to be used as a food material. For example, it can also be used directly as sugar for meals, or it can be added to foods such as fermented milk, lactic acid bacteria drinks, bread, jams, and snacks. The added concentration at this time is not particularly limited as long as it is appropriately determined in view of its flavor, physical properties, and the like. In addition to such foods, it can also be used in cosmetics, pharmaceuticals, and the like.

The following examples illustrate the invention in more detail, but the invention is not limited in any way. [Example]

Example 1 Weigh 15g of Japanese Pharmacopoeia grade lactose in a 100mL Erlenmeyer flask, and add 85g of Bis-Tris buffer (pH 6.8) (lactose concentration 15) prepared with deionized water (without sodium and magnesium ions). %). After lactose was completely dissolved in a boiling water bath, it was cooled in a constant temperature water tank at 45 ° C. 2.6 M sodium chloride was added so that the sodium ion concentration reached 15 mM, and 0.75 M magnesium chloride was added so that the magnesium ion concentration reached the concentration described in Table 1 below. To this, 600 U of 1 g of lactose was added. GODO-YNL (β-galactosidase derived from Kluyveromyces lactis, manufactured by Contract Alcohol Co., Ltd.) was added in proportions, and reacted at 40 ° C. These reaction solutions were sampled over time until the seventh hour, and the temperature was raised to 90 ° C in a boiling water bath to deactivate the enzyme, and the residual disaccharides and trisaccharides were measured by HPLC analysis under the following conditions. The ratio of galactooligosaccharides. Table 1 shows the measurement results when the amount of galacto-oligosaccharides having a trisaccharide or more at each magnesium ion concentration reached the maximum. In addition, the residual disaccharide contained unreacted lactose and transglycosidated disaccharide.

<HPLC conditions> Column: Shodex SUGAR KS-802 Mobile layer: pure water Flow rate: 0.5mL / min Detection: Differential refractometer

Example 2 The ratio of a residual disaccharide to a galacto-oligosaccharide with a trisaccharide or more was measured in the same manner as in Example 1 except that 2.6 M sodium chloride was added so that the sodium ion concentration reached 30 mM. The results are shown in Table 2.

Example 3 Depletion The concentration of lactose in the reaction solution was 45%, and 2.6 M sodium chloride was added so that the sodium ion concentration reached 5 mM. GODO-YNL was added at a rate of 250 U to 1 g of lactose, and the temperature was adjusted to 45 ° C. Except for the reaction, the ratio of the residual disaccharide to the galacto-oligosaccharide of trisaccharide or more was measured in the same manner as in Example 1. The results are shown in Table 3.

Example 4 Depletion The concentration of lactose in the reaction solution was 45%, 2.6 M sodium chloride was added so that the sodium ion concentration reached 60 mM, and GODO-YNL was added at a ratio of 250 U to 1 g of lactose, and the temperature was adjusted to 45 ° C. Except for the reaction, the ratio of the residual disaccharide to the galacto-oligosaccharide of trisaccharide or more was measured in the same manner as in Example 1. The results are shown in Table 4.

From Tables 1 and 2, it can be seen that by adding sodium ions with a concentration of 15 mM and 30 mM and magnesium ions above 0.5 mM, the reaction time before the amount of galacto-oligosaccharides with a trisaccharide or more reaches the maximum, and the concentration of magnesium ions When it is 0 mM and 0.1 mM, it is shortened to less than half. In addition, it was shown that as the concentration of magnesium ions increases, the amount of galactose oligosaccharides higher than trisaccharides also increases. In addition, it was found that when the sodium ion concentration was 15 mM, the reaction time before the maximum amount of trisaccharide or more galactooligosaccharide was produced could be further shortened by adding magnesium ions to 1.5 mM or more. As can be seen from Tables 3 and 4, when the sodium ion concentration is 5 mM and 60 mM, by adding magnesium ions to 0.5 mM or more, the galactose can be shortened by more than trisaccharides when the magnesium ion concentration is 0 mM and 0.1 mM The reaction time before the amount of oligosaccharide produced reaches the maximum; it shows that as the concentration of magnesium ions increases, especially when the concentration of magnesium ions is above 1.5 mM, the amount of galactose oligosaccharides above the trisaccharide is also increased. [Industrial availability]

According to the present invention, it is possible to increase the amount of galacto-oligosaccharides produced from trisaccharides or more with a short reaction time, and it is useful as an industrial method for producing galacto-oligosaccharides.

Claims (3)

A method for producing galactooligosaccharides, characterized in that β-galactosidase is reacted with a substrate in the presence of 5 to 60 mM sodium ions and 0.5 to 8 mM magnesium ions. The method according to claim 1, wherein the concentration of magnesium ions is 1.5 to 8 mM. The method according to claim 1 or 2, wherein β-galactosidase is derived from a microorganism belonging to the genus Kluyveromyces.